Biological cells live and communicate via complex pathways, bathing in a sea of nutrients, chemicals and cellular factors as they perform their programmed duties. In some cases of disease and infection, regiments of cells are summoned to participate or otherwise bolster the body's defense mechanisms. A variety of molecules with biological activity penetrate into the cell nucleus and bind to DNA in the double-stranded state or in the single-stranded state, thereby participating in the opening or closing of DNA helices, and hence are involved in the up or down regulation of RNA/protein synthesis. In the face of disease, cells may respond in dramatic fashion, abandoning present duties to adopt new roles, sometime differentiating to become antibody-producing or scavenger cells. Yet other cells performing routine and beneficial housekeeping tasks such as cellular repair may be subverted to produce factors that assist in the growth or vascularization of cancerous tissue.
At times, cells adjacent or distant from diseased tissue may be alerted to the presence of disease by cellular factors circulating in the system. However, instead of responding in an obvious manner, various cells may undergo a subtle reorganization of DNA, which may be observed in both condensed chromatin (heterochromatin) and more loosely-packed DNA (euchromatin) which may be currently active in RNA/protein synthesis. Typically, to observe DNA by these methods requires that DNA be visually enhanced, which is accomplished with staining. However, to measure the amount of DNA and/or the distribution of DNA requires that DNA be stained, stoichiometrically, that is, specifically and proportionately.
While changes in total DNA content (not associated with cell mitosis) have been used diagnostically, more recently, improved methods have been introduced to detect diseases, such as cancer, based upon configurational changes in the spatial distribution of DNA. One such method utilizes what are called malignancy-associated changes (“MAC”) which are sometimes observed in the presence of diseases, such as cancer, and provide one example of a generally non-specific response to cell factors.
Malignancy-associated changes (MAC) are considered to be subtle changes observed primarily in the distribution of DNA in ostensibly normal cells. Assays based on MAC differ significantly from measurements based on total DNA or genetic tests which detect specific DNA alterations, since unlike MAC, these assays rely on abnormal cells. Therefore, while malignancy-associated changes (MAC) may be less specific than genetic tests, they provide a means to detect the presence of disease, or DNA distributional changes in ostensibly normal cells. Advantages of MAC include the ability to utilized cells from associated or non-associated tissue to detect diseases such as cancer.
Associated tissue would be a sample that may reasonably be expected to contain ostensibly normal cells from the tissue being tested. For example, if lung sputum was used to screen for lung cancer, it would be considered to be an associated tissue (contains exfoliated lung cells), as would nipple aspirates for the detection of breast cancer. A discussion of nipple aspirates may be found in Petrakis, “Physiologic, Biochemical, And Cytologic Aspects Of Nipple Aspirate Fluid”, with additional discussion provided by Leif in “Centrifugal Cytology Of Nipple Aspirate Cells”. Alternatively, if a more accessible or convenient source of cells (which may have been alerted to the presence of disease via chemical messages), which is unlikely to contain a significant number of cells from the tissue under test, this would be considered to be a non-associated tissue. For example, if cells derived from the oral cavity (buccal mucosa) were used for an assay to detect lung cancer, then buccal mucosa would be considered to be a non-associated tissue.
One potential limitation of the MAC paradigm is that it is based upon staining substantially all the DNA in the cell nucleus, which may obscure cellular activity associated with RNA/protein synthesis, as contemplated by the present invention.
It would therefore be advantageous to provide a disease detection method, based on ostensibly normal cells, which offers increased sensitivity to the presence of disease. The present invention is such a method and provides a means to detect and monitor disease based on changes measured in the amount of DNA euchromatin and/or the distribution of DNA euchromatin, thus providing an indirect way of assessing a cell's current potential for RNA/protein synthesis.
As used herein, “cell response factor” (CRF) means any cell response to an outside influence such as proteins, chemicals, stress (e.g. heat, magnetic or electromagnetic energy, pressure, or other forms of energy), medication, vitamins, herbs, cosmetics, or environmental conditions, which may be measured or observed in the amount of DNA euchromatin and/or the distribution of DNA euchromatin in biological cells. While the concentration and degree of stress encountered by cells, in vivo, may be limited, the present method may be used alone or in combination with other assays or biomarkers in plant cells, cultured cells, stem cells, bacteria, or any other source of biological cells, living or dead.
Generally DNA euchromatin is that unique combination of DNA, RNA and proteins that allow the magnificent cellular program within the cell nucleus to proceed with accuracy, safety and flexibility. As used herein, “euchromatin” or “DNA euchromatin” means that portion of DNA in biological cells that appears to be transcriptionally active. This definition includes those DNA portions that are loosely coiled. Approximately 10 percent of DNA euchromatin may present in cells as 10 nm fibers, with some open strands approximately 4 nm in diameter. The balance of DNA euchromatin typically appears as a 20 to 30 nm fibers. Conversely, heterochromatin is more condensed than euchromatin, is not transcriptionally active, and may be further coiled to form fibers of in the range of 300 nm in diameter.
In biology, since the discovery of DNA and its association with diseases, such as cancer, substantial efforts have been made to develop methods to quantify the DNA content of biological cells. More recently, cytologists have been provided with tools such as image cytometers, densitometers, flow cytometers and laser scanning cytometers, to measure cell features such as size, shape, DNA content and DNA distribution. To measure total cellular DNA by image cytometry requires that DNA first be stained, stoichiometrically, that is, proportionately to the amount of DNA. The Feulgen method is one such DNA staining method and the contrast agent is often pararosaniline or a thiazine derivative. The most common stains used in Feulgen procedures include pararosaniline, azure A, thionin, and acriflavine (which may be utilized in both absorbance and fluorescent staining procedures). Additional details regarding useful DNA stains may be found in Mikel's publication entitled, “A Comparative study of quantitative stains for DNA in image cytometry”. 
To stain DNA using the Feulgen method, DNA is first hydrolyzed, typically using hydrochloric acid, which specifically and quantitatively removes purine bases, leaving the pyrimadine-sugar linkage of the DNA intact. Stripped deoxyribose sugars expose aldehyde groups along the backbone of the DNA which are subsequently coupled to Schiff's reagents to produce a staining intensity, which, ideally, is directly proportional to the amount of DNA in the cell.
Feulgen staining methods evolved over several decades and during this development a number of variables that influence DNA staining were identified. These include cell fixation, reaction temperature, hydrolysis time, acid concentration, tissue type and chromatin compactness. Two general Feulgen staining methods for DNA became accepted, differing primarily in the conditions for DNA hydrolysis. The first method advocates DNA hydrolysis at room temperature (25° C.) at a relatively high acid concentration (5 N HCL). The second adopts a reaction temperature of 60° C. using 1 N HCL.
Briefly, cells deposited on a microscope slide are immersed under the hydrolysis conditions described above, typically for between 20 and 65 minutes. During this time, ideally, all the purine bases (adenine and guanine) are removed from the DNA. This reduced state may be relatively stable over some period of time after which continued acid hydrolysis causes degradation of the DNA, as may be indicated by a decrease in optical density.
While studying DNA hydrolysis, some researchers observed that a fraction of the DNA appears to stain quickly. They called this portion of DNA, acid-labile, and began to study the kinetics of acid hydrolysis more closely, hoping to use the acid-labile characteristics to differentiate normal cells from diseased. Further discussion may be found in Sincock, “Semi-Automated Diagnosis Of Cervical Intra-Epithelial Neoplasia Grade 2 By The Measurement Of Acid Labile DNA In Cytologically Normal Nuclei”, Soames, “Feulgen hydrolysis profiles and acid-labile DNA in oral squamous cell carcinoma”, Finch, “Malignancy Associated Changes In Buccal Smears”, Klawe, “Malignancy-Associated Changes (MAC) In Cells Of Buccal Smears Detected By Means Of Objective Image Analysis”, Partington, “Quantitative Determination Of Acid-Labile DNA In Cervical Intraepithelial Neoplasia-A Potential Aid In The Diagnosis Of Malignancy”, Ogden, “The Effect Of Distant Malignancy Upon Quantitalive Cytologic Assessment Of Normal Oral Mucosa”, Sincock, “A Semi-Automated Procedure For Aiding The Diagnosis Of Cervical Neoplasms Based On The Measurement Of Acid-Labile DNA In Exfoliated Cells”, Sincock, “Semiautomated Measurement Of Rapidly Hydrolyzed DNA In The Diagnosis Of Mammary Carcinoma”, and Sincock, “Quantitative Assessment Of Cervical Neoplasia By Hydrolysed DNA Assay”. 
Unfortunately, no widely accepted assay based on the total amount of acid-labile DNA evolved from these studies. Limitations include the need to prepare multiple slides, under strict conditions, therefore increasing the time, cost and complexity of these potential methods. In addition, as will be discussed further, these applications, like MAC assays, may have reduced sensitivity to cellular changes associated with RNA/protein synthesis.
Another aspect of DNA that attracted attention was its spatial distribution in cell nuclei. In the late 1950s Nieburgs identified subtle cellular changes which he associated with disease.
When first described, malignancy-associated changes (MAC) were a curiosity. Nieburgs; “Recent Progress In The Interpretation Of Malignancy Associated Changes (MAC)”, ACTA Cytologica 1968, Vol. 12, No.6. Various researchers sought to duplicate Neiburgs' work. Only recently has the MAC paradigm resurfaced and an automated measurement method been suggested. While still controversial, MAC are described as subtle changes measured in the DNA distribution of ostensibly normal cell nuclei and are associated with non-specific and potentially systemic responses to tumor or other cell factors. Hence, the present invention adopts the general term “cell response factor” or “CRF” as convenient nomenclature.
MAC methods are described in U.S. Pat. No. 5,889,881 and further in U.S. Pat. No. 6,026,174. In addition, co-pending U.S. patent application Ser. No. 10/232,698, to MacAulay, Ferguson et al., filed on approximately Aug. 29, 2002, entitled, “Computerized methods and systems related to the detection of malignancy-associated changes (MAC) to detect cancer”, further describes methods to improve the assessment of MAC based on the normalization of digital images prior to cell feature calculation, thereby maintaining the discriminating power of these cell features. The present invention includes embodiments that support image normalization for use in the determination of a CRF or other descriptors of DNA euchromatin.
DNA euchromatin may be preferentially stained and assessed. In some instances, for example when cells are deposited on a receiving surface, such as a microscope slide, the same cells may be further hydrolyzed and the remaining DNA stained. This provides a means to compare the amount and distribution of DNA euchromatin with total cellular DNA and its distribution, on a cell-by-cell basis. The amount as well as the location sites of DNA synthesis measured in this manner may provide additional diagnostic information. These abilities may have increased importance as other microscopic methods gain broader acceptance. For example, confocal microscopy provides the ability to collect a plurality of cellular image slices for three dimensional reconstruction. (e.g. U.S. Pat. No. 6,388,809) The caveat regarding receiving surface serves as a reminder that other measurement tools, such as flow cytometers, may provide the ability to measure DNA and related cellular features as contemplated, herein. These systems maintain cells in a fluid environment, typically directing them to a sensor that often includes a laser. After analysis by flow cytometry, unless special efforts, such as cell sorting, are utilized, the biological cells used in the assay are typically lost.
Decades of effort has gone into optimizing Feulgen methods to stain substantially all DNA and although Feulgen methods do not typically provide for preferentially staining DNA euchromatin, they do offer a step in the process which may be exploited to advantage for the present invention. This step relates to DNA hydrolysis, whereby acid is used to selectively and specifically strip away purine bases from the DNA backbone. Typically a DNA absorbance stain such as pararosaniline or a thiazine derivative such as thionin is used as a contrast agent. Other DNA stains include propidium iodide, adenine-thiamine selective stains such as DAPI, Hoechst (33342 and 33258), SYTOX (blue, green or orange), and cytosine-guanine stains such as chromomycin A3 and mithramycin. These stains, however, do not typically differentiate between dense chromatin and euchromatin. However, in future it may be possible to block certain DNA sites and subsequently exploit one or more of these DNA fluorescent stains for the present invention. New methods to rapidly stain nucleic acids (e.g. U.S. Pat. No. 6,271,035) are being introduced and microwaves have also been employed to facilitate staining.
Another method to assess DNA and DNA euchromatin is based on the methylated state of DNA. These techniques are relatively complex and typically require that DNA be removed from cells and be further manipulated using PCR or other techniques. Again, DNA methods based on methylated state function optimally on abnormal cells which may be difficult to obtain or access, sometimes requiring a biopsy procedure.
It would therefore be advantageous to provide a simple method of detecting and monitoring disease that could be applied to readily accessible, ostensibly normal cells. While tissue obtained from biopsies may be employed for the present invention, for high volume applications, such as, at risk population screening, it may be preferable to use scraping of cells from accessible body cavities (e.g cervix), body fluids (e.g. blood or urine), aspirates (e.g. breast or fine needle), washings (e.g. bronchial-lavage or bladder washings), or samples typically rich in exfoliated cells (e.g. lung sputum or cells from the oral cavity). The present method may also prove useful for detecting or monitoring disease where specific markers or disease mechanisms are not yet fully understood, for example, auto-immune diseases, stress disorders, chronic fatigue syndrome, allergies, age related disorders, infections, or other degenerative diseases such as Alzheimer's. Similarly, the complex interaction of vitamins, herbs, food supplements, medications and exposure to various forms of energy from sunlight to cell phones may also cause cellular changes observable in DNA euchromatin. These may be utilized, for example, in monitoring a diseases response to treatment, assessing wellness, evaluating bio-activity or screening pharmacological agents. Generally speaking, anything that causes or is associated with changes in RNA/protein synthesis in cells is of potential interest, whether these changes occur in microorganisms, plants or humans. In addition, it may be useful to use DNA euchromatin assessments to further characterize cell death (apoptosis) or other biological processes, or as a basis for new assays, such as determination of ‘time of death’ by sampling dead or dying cells, for example.
In the mid 1960s, Decosse and Aiello published the paper entitled “Feulgen Hydrolysis: Effect Of Acid And Temperature” describing DNA acid hydrolysis and concluding that Feulgen hydrolysis at room temperature (26° C.) using 5.0N HCL was essentially equivalent DNA acid hydrolysis at 60° C. using 1.0N HCL. The authors noted that the 120-minute plateau provided by the former at room temperature was superior to the 20-minute stability observed at 60° C. Additionally, they concluded that depurination (removal of purine bases from DNA) depended primarily on acid concentration and that subsequent DNA degradation is dependent primarily on heat rather than acid. Accordingly, the reaction temperature and conditions for hydrolysis go against what is discussed in prior art. Some embodiments of the present invention identify DNA euchromatin for assessment by preferential staining, for example, lowering the reaction temperature for acid hydrolysis of DNA by approximately 10 degrees C. appears to slow hydrolysis (at a given acidity) four fold, thus providing improved control over DNA staining and more particularly facilitates preferential staining of DNA euchromatin
Later, Fukuda summarized the history of DNA acid hydrolysis and staining in “Errors in Absorbance Cytophotometry” with additional discussion in “Biological Application Of Absorbance Cytophotometry”. Similar conclusions were observed by Zelenin in “Peculiarities Of Cytochemical Properties Of Cancer Cells As Revealed By Study Of Deoxribonucleoprotein Susceptibility To Feulgen Hydrolysis” and Kjellstand in “Temperature And Acid Concentration In The Search For Optimum Feulgen Hydrolysis Conditions”. 
More recently, U.S. Pat. No. 5,016,283, to Bacus, entitled, “Methods and apparatus for immunoploidy analysis”, teaches acid hydrolysis for 60 to 75 minutes in 5 N HCL followed by thionin staining for 60 minutes. Similarly, U.S. Pat. No. 5,485,527, to Bacus, entitled, “Apparatus and method for analysis of biological specimens”, teaches DNA acid hydrolysis in 5 N HCL for 60 to about 75 minutes. And U.S. Pat. No. 5,942,410, to Lam, entitled, “Composition and method for staining cellular DNA, comprising thiazine derivative metabisulfite and method”, summarizes the prior art for DNA staining and further promotes a DNA hydrolysis time of 60 minutes in 5 N hydrochloric acid followed by 75 minutes of staining. DNA staining is also discussed in U.S. Pat. No. 6,348,325, to Zahniser, entitled “Cytological stain composition.”
Current devices and methods to measure and exploit DNA measurements are taught in U.S. Pat. No. 5,889,881, to MacAulay, entitled, “Method and apparatus for automatically detecting malignancy-associated changes”, and also U.S. Pat. No. 6,026,174, to Palcic, entitled, “System and method for automatically detecting malignant cells and cells having malignancy-associated changes”. This prior art teaches both the use of DNA content (ploidy) and MAC (subtle changes reflected primarily in the distribution of DNA within ostensibly normal cells) for disease detection as well as discussing a variety of useful cell features. In addition, DNA descriptors for disease detection using MAC are further discussed in co-pending U.S. patent application Ser. No. 10/232,698, to MacAulay, Ferguson et. al., filed on approximately Aug. 29, 2002, entitled, “Computerized methods and systems related to the detection of malignancy-associated changes (MAC) to detect cancer”, which among other things discusses DNA measurements, cellular features and methods to normalize cell features by first normalizing the digital images of cells.
This MAC prior art also discusses utilizing combinations of cellular features and reducing DNA measurements to a value, such as MAC score, which like CRF, may be considered to be a cell response factor. Accordingly, this MAC prior art and other prior art cited in this application are included by reference, herein.
Today it is understood that DNA helices must undergo localized strand separation at particular gene loci for the onset of RNA or DNA synthesis. Such activity is observed during both gene transcription and replication. By far, the vast majority of DNA in various cell types remains inactive, after cellular programming. Accordingly, it may be useful to measure both total DNA and DNA euchromatin and express these values as a ratio, such as percent DNA euchromatin.
In related studies, investigators considered that an increase in acid-labile DNA may be associated with malignancy. Acid-labile DNA was explored by Sincock. He suggested lower hydrolysis temperatures than Fukuda (“Errors In Absorbance Cytophotometry”) and performed hydrolysis at 30° C. in 5 N HCL indicating that certain diseases (CIN 2) may cause increase the percentage of cells in S-phase (the phase during which cells in mitosis copy substantially al of their DNA). Previously, Millett in “Feulgen-Hydrolysis Profiles In Cells Exfoliated From The Cervix Utero. A Potential Aid In The Diagnosis Of Malignancy” also suggested lower temperatures than those used in previous studies and opted for 5 N HCL at room temperature. Similarly with, Partington made this suggestion in “Quantitative determination of acid-labile DNA in cervical intraepithelial neoplasia”. In Soames' 1995 paper entitled “Feulgen Hydrolysis Profiles And Acid-Labile DNA In Oral Squamous Cell Carcinoma”, hydrolysis conditions were 5 N HCL at room temperature. While Kjellstand, cited above, discusses a wide range of temperature and acidity for Fuelgen hydrolysis, he does not discuss or acknowledge advantages associated with partial DNA staining and accordingly provides conclusions and recommendations that go against the methods and embodiments of the present invention.
U.S. Pat. No. 5,871,917, to Duffy, entitled, “Identification of differentially methylated and mutated nucleic acids”, among other things, discusses methods for detecting and isolating genomic DNA fragments that are near coding and regulatory regions of genes. It is noted that DNA is frequently methylated in tumor cells.
U.S. Pat. No. 6,451,555, to Duffy, entitled, “Nucleic acids that encode testes specific protease and detect DNA hypomethylated in cancer cells”, discusses methods for detecting and isolating genomic DNA fragments which are near coding and regulatory regions of genes and sensing the extent that DNA is methylated in various regions.
U.S. Pat. No. 5,556,750, to Modrich, entitled, “Methods and kits for fractionating a population of DNA molecules based on the presence or absence of a base-pair mismatch utilizing mismatch repair systems”, discusses contacting and comparing DNA strands to detect base pair mismatches using DNA protein complex formation as an indicator.
While the human genome project addressed essentially a linear problem (nucleic acid sequences), proteins present a three-dimensional problem deriving much of their functionality from shape and exposed or charged regions that allow them to react and interact with other chemicals and proteins, often with high specificity. Accordingly, while the present invention does not seek to measure specific aberrations, such as base-pair mismatch, it does seek to measure changes related to RNA/protein synthesis at a cellular level.
U.S. Pat. No. 5,206,244, to Zahler, entitled, “Hydromethyl (methylenecyclopentyl) purines and pyrimidines”, discusses methylenation reagents and various factors related to protein synthesis and more particularly methylated state of the building blocks of DNA—the purines, adenine and guanine, and pyrimadines, cytosine and thymine.
U.S. Pat. No. 5,936,064, to Baxter, entitled “Acid-labile subunit (ALS) of insulin-like growth factor binding protein complex”, discusses a specific acid-labile protein and its fragments. This prior art relates generally to acid-labile proteins and means to assess proteins states based on their amino acids.
U.S. Pat. No. 5,643,556, to Gilchrest, entitled “Stimulation of tanning by DNA fragments or single-stranded DNA”, among other things discusses damage to skin from exposure to agents such as ultraviolet light. While this patent (556) is interested in melanogenesis-stimulation, the present invention could be used to help assess if various agents are associated with a cellular response.
U.S. Pat. No. 5,773,219, to Sanford-Mifflin, entitled “Process for detecting Alzheimer disease using cultured cells”, uses DNA assessments such as gaps and breakage. Such changes may also be inferred by DNA euchromatin assessments as contemplated herein.
U.S. Pat. No. 5,670,621, to Donahue, entitled, “DNA structure specific recognition protein complexes”, among other things discusses DNA structure, a mammalian cellular factor and drug responses.
U.S. Pat. No. 6,455,593, to Grimley, entitled “Method of dynamic retardation of cell cycle kinetics to potentiate cell damage”, describes cellular restraining agents and targeted cytotoxic insults. Again, the present invention may be used independently or in combination to help assess or guide discovery of various agents.
U.S. Pat. No. 6,391,026, to Hung, entitled, “Methods and systems for treating breast tissue”, describes diagnostic methods and energy forms used to treat breast disease. The present invention could be used for example to assist in monitoring the effectiveness of such treatment.
U.S. Pat. No. 6,287,521, to Quay, entitled “Devices and methods for obtaining and assaying mammary fluid samples for evaluating breast diseases, including cancer”, discusses obtaining biological samples containing cells, such as mammary fluid. As described herein, breast aspirates would be considered an associated tissue for detecting breast cancer as contemplated by the present invention.
U.S. Pat. No. 6,035,258, to Freed, entitled, “Method for correction of quantitative DNA measurements in a tissue section”, further discusses Feulgen staining of histological tissue. As desired, similar methods could be applied to the present method.
U.S. Pat. No. 5,989,816, to Van Houten, entitled, “Method to detect DNA damage and measure DNA repair rate”, discusses DNA assays to measure DNA repair and monitor the efficacy of various therapies. The present invention may be used to support such assays.
U.S. Pat. No. 5,633,945, to Kamentsky, entitled, “Accuracy in cell mitosis analysis” describes DNA staining with fluorescent stains and measurement using a cytometer. Accordingly, DNA measurements and assessment of the cell cycle are discussed and plotted as for example in FIGS. 3, 14, 15 and 16 of that patent. Accordingly, DNA histograms are represented in as prior art in FIG. 1a of the present invention.
U.S. Pat. No. 6,215,892, to Douglass et. al., entitled, “Method and apparatus for automatic image analysis of biological specimens”, discusses an image cytometer, which may be used to measure DNA.
U.S. Pat. No. 5,849,595, to Alfano, entitled, “Methods for monitoring the effects of chemotherapeutic agents on neoplasmic media”, among other things, discusses agents such as retinoic acid and means to gauge effects at a cellular level. Monitoring the effects of various agents is contemplated for the present invention.
U.S. Pat. No. 3,957,583, to Gibson, entitled, “Apparatus and process for determining the susceptibility of microorganism to antibiotics”, discusses some of the issues and interests in assessing bio-activity and pharmacological screening as well as various culture media.
U.S. Pat. No. 5,016,283, to Bacus, entitled, “Methods and apparatus for immunoploidy”, discusses another configuration of image cytometer as well as methods to stain and measure DNA (e.g. Feulgen), sometimes in conjunction with other bio-indicators, such as estrogen. In addition, this patent (283) discusses various ways of expressing DNA content, for example, using a DNA calibrator and converting DNA values to picograms. As desired such conversion could be applied to DNA euchromatin as contemplated herein.
U.S. Pat. No. 5,485,527, to Bacus, entitled, “Methods and apparatus for analysis of biological specimens” further discusses DNA measurements, providing yet another example of established and suggested DNA staining (e.g. using thionin) and more particularly DNA hydrolysis conditions.
DNA staining is discussed in further detail in U.S. Pat. No. 5,168,066, to Zahniser, entitled, “Thionin staining and imaging technique”, and further discusses DNA staining with thionin as well as counter staining various cellular components such as the cytoplasm.
U.S. Pat. No. 5,942,410, to Lam, entitled, “Composition and method for staining cellular DNA, comprising thiazine derivative metabisulfite and methanol or ethanol”, further discusses DNA and Feulgen staining methods.
U.S. Pat. No. 5,862,304, to Ravidin, entitled, “Method for predicting the future occurrence of clinically occult or non-existent medical conditions”, discusses data evaluation and DNA histograms for prognosis.
U.S. Pat. No. 6,454,705, to Cosentino, entitled, “Medical wellness parameters management system, apparatus and method”, discusses patient monitoring and refining information to form a score, as well as discussing recognition of trends and monitory frequency. While this patent (705) describes a systematic decision making process to identify symptomatic patients, in addition, the present method may be used to identify non-specific changes (e.g. changes in RNA/protein synthesis) observed in DNA euchromatin which may be used for screening asymptomatic groups, such as current or past smokers for lung related diseases, including cancer.